Cellular senescence is characterized by a "dominant" phenotype whereby cells are no longer able to proliferate. Although little is known about the mechanisms involved in inducing/mediating cell senescence, recent studies have demonstrated important alterations in components of signal transduction and cell regulatory pathways in cell senescence. Many of these changes appear to be of a "recessive" nature and no distinction has been made between primary and secondary effects: none-the-less, these results are pointing to the importance of studying the regulation and operation of these pathways in cell senescence as an integral component of any biochemical and molecular understanding of the mechanisms of cell senescence (in analogy with the current understanding of cancer molecular biology). In particular, signal transduction pathways (such as the phosphatidylinositol cycle) are emerging as critical regulators of cell growth and differentiation. The sphingomyelin cycle is a recently described pathway that plays an important role in cell regulation. Ceramide, the product of sphingomyelin hydrolysis, has assumed a critical role as a novel second messenger that mediates differentiation and apoptosis in lymphocytes and leukemia cells. Exciting data from the PI's laboratory demonstrate that ceramide levels are significantly elevated in cellular senescence. Additional results are beginning to implicate ceramide as a "dominant" inducer of senescence. A very recent and important discovery by the PI demonstrates that phospholipase D is inhibited in senescence resulting in lack of diacylglycerol generation and protein kinase C activation. Furthermore, we show that ceramide inhibits this phospholipase D/diacylglycerol/protein kinase C pathway in young fibroblasts; thus recapitulating this senescence defect. Therefore the current proposal is based on the hypothesis that ceramide induces the senescent phenotype, and that it does so, at least in part, by inhibiting the phospholipase D mitogenic pathway. The specific aims of this proposal are l) to determine the role of ceramide in inducing the senescent phenotype by: A) determining if ceramide induces a senescent morphology; B) determining ceramide levels in cell cycle of young HDF; C) determining if ceramide can induce cell cycle arrest, and D) examining if ceramide can upregulate the senescence derived inhibitor (sdi1). 2) To determine the mechanism and source of ceramide elevation in cellular senescence by: A) determining if sphingomyelin is the source of ceramide; B) determining if glycolipids are additional sources of ceramide; C) measuring de novo synthesis of ceramide, and D) evaluating ceramide catabolism. 3) To determine the mechanism by which ceramide induces the senescent phenotype. This specific aim will concentrate on the ability of ceramide to suppress the phospholipase D/diacylglycerol/protein kinase C mitogenic pathway by determining: A) if ceramide blocks c-fos induction and AP1 activation; and B) the mechanism by which phospholipase D is inhibited in senescent cells and by ceramide in young cells, by studying this enzyme in permeabilized cells and in reconstituted cellular fractions. These studies will provide unique insight into the role of these lipid signal transduction pathways in cellular senescence and will allow better understanding of the molecular mechanisms involved in this important developmental program.